Peptide therapeutic agents are well known and are of increasing use in the pharmaceutical arts. Hormones, immunomodulators, and a host of newly discovered peptide and peptide-like compounds including certain immunogens are presently being administered to animals, including humans, in therapeutic regimens.
Consistent drawbacks to the parenteral administration of such peptide compounds have been the rapidity of breakdown or denaturation (loss of "native state configuration") of such compounds in the physiological environment and the difficulty of obtaining therapeutically effective dosage levels of such agents for extended periods. Infusion pumps, as well as wax or oil implants, have been employed in the therapeutic arts for chronic administration of therapeutic agents in an effort to both prolong the presence of peptide-like therapeutic agents and preserve the integrity of such agents. Furthermore, in particular cases in which the peptide-like therapeutic agent (which will be understood to include a protein or haptene) is to function as an immunogen, the peptide-like agent should (with particular reference to each epitope of the peptide-like agent) ideally maintain native state configuration for an extended period of time and additionally be presented in a fashion suitable for triggering an immunogenic response in the challenged animal.
One adaptation of the administration of peptide-like therapeutic agents is in the vaccine art. In this art immunogens are introduced into an organism in a manner so as to stimulate an immune response in the host organism. The induction of an immune response depends on many factors among which are believed to be the chemical composition and configuration of the immunogen, the immunogenic constitution of the challenged organism, and the manner and period of administration of the immunogen. An immune response has many facets, some of which are exhibited by the cells of the immune system, (e.g.,B-lymphocytes, T-lymphocytes, macrophages, and plasma cells). Immune system cells may participate in the immune response through interaction with immunogen or other cells of the immune system, the release of cytokines and reactivity to those cytokines. Immune response is conveniently (but arbitrarily) divided into two main categories--humoral and cell-mediated. The humoral component of the immune response includes production of immunoglobulins specific for the immunogen. The cell-mediated component includes the generation of delayed-type hypersensitivity and cytotoxic effector cells against the immunogen.
In some instances the immune response is the result of an initial or priming dose of an immunogen that is followed by one or more booster exposures to the immunogen. Priming with relatively strong immunogens and liposomes is discussed in "Liposomal Enhancement of the Immunogenicity of Adenovirus Type 5 Hexon and Fiber Vaccines", Kramp, W. J. et al., Infection and Immunity, 25:771-773 (1979) and "Liposomes as Adjuvants with Immunopurified Tetanus Toxoid: the Immune Response", Davis, D. et al., Immunology Letters, 14:341-8 (1986/1987).
Ideally, an immunogen will exhibit two properties; the capacity to stimulate the formation of the corresponding antibodies and the propensity to react specifically with these antibodies. Immunogens bear one or more epitopes which are the smallest part of an immunogen recognizable by the combining site of an antibody.
In particular instances, immunogens, fractions of immunogens or conditions under which the immunogen is presented are inadequate to precipitate the desired immunological response. Insufficient immunity occurs as a result. This is often the case with peptides or other small molecules used as immunogens. Other substances such as immunomodulators (e.g., cytokines such as the interleukins) may be combined in vaccines as well.
The vaccine art recognizes the use of certain substances called adjuvants to potentiate an immune response when used in conjunction with an immunogen. Adjuvants are further used to elicit an immune response that is faster or greater than would be elicited without the use of the adjuvant. In addition, adjuvants may be used to create an immunological response using less immunogen than would be needed without the inclusion of adjuvant, to increase production of certain antibody subclasses that afford immunological protection or to enhance components of the immune response (e.g., humoral, cellular). Known adjuvants include Freund's Adjuvants (and other oil emulsions), Bordetella Pertussis, aluminum salts (and other metal salts), Mycobacterial products (including muramyl dipeptides), and liposomes.
As used herein, the term "adjuvant" will be understood to mean a substance or material administered together or in conjunction with an immunogen which increases the immune response to that immunogen. Adjuvants may be in a number of forms including emulsions (e.g., Freund's adjuvant), gels (aluminum hydroxide gel), particles (liposomes) or solid materials.
It is believed that adjuvant activity can be effected by a number of factors. Among such factors are (a) carrier effect, (b) depot formation, (c) altered lymphocyte recirculation, (d) stimulation of T-lymphocytes, (e) direct stimulation of B-lymphocytes and (f) stimulation of macrophages.
With many adjuvants, adverse reactions are seen. In certain cases, adverse reactions may include granuloma formation at the site of injection, severe inflammation at the site of injection, pyrogenicity, adjuvant induced arthritis or other autoimmune response, or oncogenic response. Such reactions have hampered the use of adjuvants such as Freund's adjuvant. The search continues for additional adjuvants that promote immunogenic activity yet are non-toxic to the host.
In particular embodiments of the present invention, liposomes are utilized as adjuvants either alone or in combination with other adjuvants. U.S. Pat. No. 4,053,585 issued Oct. 17, 1977 to Allison et al. states that liposomes of a particular charge are adjuvants. Immunogenic compositions according to the present invention are clearly distinguishable over this reference in both the composition and the magnitude of the immunogenic response associated with the composition. Other substances such as immunomodulators (e.g., cytokines such as the interleukins) may be combined in adjuvants as well. Davis, D, et al., "Liposomes as Adjuvants with Immunopurified Tetanus Toxoid: Influence of Liposomal Characteristics", Immunology, 61:229-234 (1987); and Gregoriadis, G. et al., "Liposomes as Immunological Adjuvants: Antigen Incorporation Studies", Vaccine, 5:145-151 (1987) report DMPC/cholesterol liposomes (in a weight ratio of 1:1) and immunogen as giving minimally improved (over free immunogen) immunological response in small unilamellar vesicles of a distinct dehydration/rehydration type with tetanus toxoid as the immunogen, a strong immunogen. In the Davis and in the Gregoriadis papers, the liposomal immunogenic response was only minimally distinguishable from the response of free immunogen. To distinguish the liposomal from free immunogen response, the authors found it necessary to dilute the tetanus toxoid to minimal response amounts. As distinguished from these references, the present invention adopts conditions of DMPC/cholesterol liposomes that yield therapeutically effective immunological response not taught by the prior art. The immunological responses produced by the dosage forms of the present invention are a surprising result. More importantly, the present invention teaches the use of DMPC/cholesterol liposomes as adjuvants for immunogenic agents that generally produce a weak immunogenic response or no immunogenic response.
Humoral immune response may be measured by many well known methods. Single Radial Immunodiffusion Assay (SRID), Enzyme Immunoassay (EIA) and Hemagglutination Inhibition Assay (HAI) are but a few of the commonly used assays of humoral immune response. SRID utilizes a layer of a gel such as agarose containing the immunogen being tested. A well is cut in the gel and the serum being tested is placed in the well. Diffusion of the antibody out into the gel leads to the formation of a precipitation ring whose area is proportional to the concentration of the antibody in the serum being tested. EIA, also known as ELISA (Enzyme Linked Immunoassay), is used to determine total antibodies in a sample. The immunogen is adsorbed to the surface of a microtiter plate. The test serum is exposed to the plate followed by an enzyme linked immunoglobulin, such as IgG. The enzyme activity adherent to the plate is quantified by any convenient means such as spectrophotometry and is proportional to the concentration of antibody directed against the immunogen present in the test sample. HAI utilizes the capability of an immunogen such as viral proteins to agglutinate chicken red blood cells (or the like). The assay detects neutralizing antibodies, i.e. those antibodies able to inhibit hemagglutination. Dilutions of the test serum are incubated with a standard concentration of immunogen, followed by the addition of the red blood cells. The presence of neutralizing antibodies will inhibit the agglutination of the red blood cells by the immunogen.
Tests to measure cellular immune response include determination of delayed-type hypersensitivity or measuring the proliferative response of lymphocytes to target immunogen.
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic "head" orient towards the aqueous phase.
The original liposome preparation of Bangham, et al. (J. Mol. Biol., 1965, 13:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys. Acta., 1968, 135:624-638), and large unilamellar vesicles. Small unilamellar vesicles have a diameter of about 100 nm or less.
Unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT Application No. WO 87/00238, published Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles" incorporated herein by reference. Vesicles made by this technique, called LUVETs, are extruded under pressure once or a number of times through a membrane filter.
A subclass of multilamellar vesicles are liposomes which are characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Pat. No. 4,558,578 to Fountain, et al. and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies." U.S. Pat. No. 4,721,612 to Janoff et al. describes steroidal liposomes for a variety of uses. The teachings of these references as to preparation and use of liposomes are incorporated herein by reference.